Expression, biochemical characterization, and mutation of a water forming NADH: FMN oxidoreductase from Lactobacillus rhamnosus.

Enzyme-catalyzed cofactor regeneration is a significant approach to avoid large quantities consumption of oxidized cofactor, which is vital in a variety of bioconversion reactions. NADH: FMN oxidoreductase is an ideal regenerating enzyme because innocuous molecular oxygen is required as an oxidant. But the by-product H2O2 limits its further applications at the industrial scale. Here, novel NADH: FMN oxidoreductase (LrFOR) from Lactobacillus rhamnosus comprised of 1146 bp with a predicted molecular weight of 42 kDa was cloned and overexpressed in Escherichia coli BL21 (DE3). Enzyme assay shows that the purified recombinant LrFOR has both the NADPH and NADH oxidation activity. Biochemical characterizations suggested that LrFOR exhibits the specific activity of 39.8 U·mg-1 with the optimal pH and temperature of 5.6 and 35 °C and produces H2O instead of potentially harmful peroxide. To further study its catalytic function, a critical Thr29 residue and its six mutants were investigated. Mutants T29G, T29A, and T29D show notable enhancement in activities compared with the wild type. Molecular docking of NADH into wild type and its mutants reveal that a small size or electronegative of residue in position29 could shorten the distance of NADH and FMN, promoting the electrons transfer and resulting in the increased activity. This work reveals the pivotal role of position 29 in the catalytic function of LrFOR and provides effective catalysts in NAD+ regeneration.

Role of introduced surface cysteine of NADH oxidase from Lactobacillus rhamnosus.

Cysteine, a critical residue for catalytic process but also vulnerable to oxidative damage, was conventionally expressed as a buried catalytic site in most redox enzymes. In the present work, specific surface-exposed sites of a NADH oxidase from Lactobacillus rhamnosus (LrNox) were selected and mutated to cysteine to investigate its effects on catalytic function because LrNox has a buried catalytic cysteine but no surface-exposed one. The results showed that exception of the sites on dimer interface, the activities of LrNox mutants were improved to vary degrees when the polar uncharged and alanine residues were mutated to cysteine. But the cysteine mutations of polar charged and nonpolar residues except alanine showed obvious decline in catalytic activity. Substituting of Ala85 and Thr96 with other residues suggested that the cysteine mutation showed the highest activity. Structural analysis suggested that even a single cysteine mutation on the specific non-conserved surface area of LrNox could induce changes on the conformation of catalytic cysteine and lower the activation free energy to improve the catalytic activity.

Immobilization of Lipase by Adsorption Onto Magnetic Nanoparticles in Organic Solvents.

In order to improve the performance of lipase in organic solvents, a simple immobilization method was developed by adsorption of lipase onto Fe3O4@ SiO2magnetic nanoparticles in organic solvent. Among the solvents tested, toluene was found to be the most effective solvent for the immobilization. A maximum immobilization yield of 97% and relative activity of 124% were achieved in toluene at 30 °C. The optimal temperature, enzyme loading and water activity were 30 °C, 1.25 mg/mg support and 0.48 aw, respectively. The residual activity of immobilized lipase was 67% after 10 cycles of use. The advantages of the immobilized lipase including easy recovery, high stability, and enhanced activity of immobilized lipase in organic solvents show potential industrial applications in anhydrous solvents.

Application of Molecular Imprinted Magnetic Fe3O4@SiO2 Nanoparticles for Selective Immobilization of Cellulase.

Magnetic Fe3O4@SiO2 nanoparticles were prepared with molecular imprinting method using cellulase as the template. And the surface of the nanoparticles was chemically modified with arginine. The prepared nanoparticles were used as support for specific immobilization of cellulase. SDS-PAGE results indicated that the adsorption of cellulase onto the modified imprinted nanoparticles was selective. The immobilization yield and efficiency were obtained more than 70% after the optimization. Characterization of the immobilized cellulase revealed that the immobilization didn't change the optimal pH and temperature. The half-life of the immobilized cellulase was 2-fold higher than that of the free enzyme at 50 degrees C. After 7 cycles reusing, the immobilized enzyme still retained 77% of the original activity. These results suggest that the prepared imprinted nanoparticles have the potential industrial applications for the purification or immobilization of enzymes.

[Hospital-based psychological first aid provided to patients injured in the Lushan earthquake].

In the aftermath of the 7.0 earthquake that struck Lushan in China's Sichuan Province on April 20, 2013, a psychological crisis intervention working group was established in a hospital that was treating earthquake victims. Patients at this hospital received psychological first aid that was delivered in accordance with scientific, systematic, and standardized principles. This first aid employed a "rooting mode" methodology and was designed as a supportive psychological intervention. Mental assessment results showed that the general mental health, acute stress reactions, and anxiety and depression status of all of the 131 injured who received the psychological intervention had significantly improved (p < .05) during the two-week intervention period. This paper introduces the basic principles used to develop and provide this first aid, the approach used to organize the working groups, the main contents of the intervention, specific methods used, and intervention outcomes. This information is provided as a reference for providing localized psychological assistance in the aftermath of a disaster incident.

[The effect of equilibrium psychological intervention in wounded patients in Lushan earthquake].

OBJECTIVE: To investigate the early psychological effect of the equilibrium psychological intervention on the person who were injured in the disaster incident. METHODS: The equilibrium psychological intervention was used to the injured person in the Lushan earthquake during the early period. The GHQ-12, HAMA and HAMD were used before and after the evaluation. RESULTS: The score of the GHQ-12 decreased from (3.488 +/- 2.900) to (1.610 +/- 0.840), which showed the significant differences (P < 0.001). The total score of the HAMA and the score of the somatic anxiety factor and mental anxiety factor decreased significantly, compared with the base line (P < 0.001 respectively). The total score of the HAMD and the score of the sretardation factor, somatization factor and sleep disorder factor also decreased significantly, compared with that of the base line (P < 0.005 respectively). CONCLUSION: The equilibrium psychological intervention has the positive effect on the persons who were injured in the disaster incident during the early period.